Regional anesthesia in faciomaxillary and oral surgery.

Faciomaxillary and oral surgical procedures are frequently done under local anesthesia. Only few techniques are used widely in these areas in spite of the numerous blocks available. Knowledge about these techniques could encourage use of these techniques for the benefit of patients and operators' comfort. Leaving aside the commonly used intraoral anesthetic technique by faciomaxillary and dental surgeons, focus is given on regional blocks of extraoral route, like maxillary block, mandibular block, superficial cervical plexus block, forehead and scalp block, trigeminal nerve block, sphenopalatine nerve block, and they are discussed with their indications and technical details involved in administering them. Advantages of using the regional blocks over general anesthesia and multiple pricks include reduced dosage and number of needle pricks. Pediatric considerations like prolonged duration of anesthesia and wider area of action for regional blocks warrant that they should be used with caution.

Development of morulae from the oocytes of cultured sheep preantral follicles.

Sheep preantral follicles (PFs) measuring 250-400 microm in diameter were cultured for six days in serum-free media supplemented differently with growth factors and hormones. Subsequently, oocytes from the cultured follicles were subjected to an additional 24 h of in vitro maturation (IVM) followed by in vitro fertilization (IVF) and embryo culture for 6 days. Five different experiments were conducted. In the first experiment individual concentrations of Insulin-Transferrin-Selenite (ITS), Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta (TGF-beta), Insulin (INS), and Growth hormone (GH) that supported the best in vitro development of the PFs were determined. The influence of different combinations of the above hormones and growth factors at their best concentrations as determined in the first experiment was investigated in the second experiment. In the third experiment the best combinations of the growth factors and hormones obtained in the second experiment were additionally supplemented with Thyroxin (T4) and follicle stimulating hormone (FSH) and the influence on in vitro development of the PFs was studied. In the fourth experiment, two methods of culturing PFs-micro drops and agar gel embedding-were compared. In the fifth experiment oocytes from cultured PFs were subjected to IVF and in vitro development of the resulting embryos was followed to the blastocyst stage. Based on the proportion of the PFs exhibiting growth, mean increase in diameter, proportions of PFs developing antrum, ovulations in vitro and oocytes maturing to M-II stage, 1% ITS, 10 ng/mL each of IGF-I, and Insulin and 1 mIU/mL of GH were found to support the best development of sheep PFs. However, the oocytes from PFs cultured in any concentration of TGF-beta failed to mature to M-II stage. Similarly, among the combinations studied, IGF-I+GH was found to be the best. In combination with T4 and FSH, IGF-I+GH supported the best development of the PFs. Culture of PFs in micro drops or agar gel supported similarly high development. In vitro fertilization of the oocytes from the cultured sheep PFs resulted in the embryos developing to the morula stage for the first time.

Controlled release of 2, 3 desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting E-selectin.

Control of inflammation using appropriate anti-inflammatory agent prevents wound from becoming chronic. Heparin is a heterogeneous mixture of polysaccharide molecules with a mean molecular weight between 12-30 kDa containing 200-300 disaccharide units of glycosaminoglycan chains. Chemical modifications leading to generation of a unique pentasaccharide sequence effectively reduces its anticoagualant activity, while retaining its anti-inflammmatory property. In this study, Standard heparin was partially desulfated to 2, 3 desulfated heparin (2, 3 DSH) and its anti-inflammatory property was determined by assessing its ability to prevent static adhesion of leukocytes to endothelium and clotting assay. The effectiveness of 2, 3 DSH to down regulate E-selectin and key proinflammatory cytokines (IL-1beta and IL-6) was assessed by western blot and immunocytochemistry. These results were compared with commercially available 2-Desulfated Heparin (2DSH) and standard heparin (SH). Finally, a controlled delivery system of 2, 3 DSH was designed using chitosan microspheres and collagen as scaffold. Optimal loading of 2, 3 DSH was achieved and the release kinetics were tuned to follow a controlled release pattern. The steady state concentration of 2, 3 DSH was found to be optimal to elicit anti-inflammatory property and could achieve inhibition of E-selectin expression while unaffecting the normal clotting cascade.

Functionally modified gelatin microspheres impregnated collagen scaffold as novel wound dressing to attenuate the proteases and bacterial growth.

An attempt was made to develop a new therapeutic delivery system which would play a dual role of attenuating MMP activity in the wounds and also prevent infection by controlled delivery of antimicrobials. A catechol type MMP inhibitor 2,3-dihydroxybenzoic acid (DHBA) was conjugated to gelatin microspheres using EDC/NHS as coupling agents. The potential of the modified gelatin microspheres (DHB-MS) to attenuate the proteases such as MMP 2 and MMP 9 in the diabetic wound tissues was investigated by gelatin zymography. Further the modified microspheres were loaded with doxycycline and impregnated in a reconstituted collagen scaffold as novel wound dressing. The in vitro release behavior of doxycycline from both DHB-MS and DHB-MS impregnated collagen scaffold was investigated. DHB-MS when incubated with the tissue lysate for 6h displayed the complete inhibition of the MMPs in the tissue lysate. The in vitro drug release studies from the collagen scaffold exhibited the burst release of 44%, exerted immediate chemo prophylaxis and sustained delivery for 72 h. The MTT assay and fluorescent labeling with calcein AM indicated that the DHB-MS is biocompatible to human foreskin fibroblasts. Thus the system developed provides wider scope to control the pathogens involved in infection and also the excess matrix degradation.

Efficiency of controlled topical delivery of silver sulfadiazine in infected burn wounds.

The present study is designed to assess the potential benefits of controlled delivery of silver sulfadiazine from collagen scaffold (SSDM-CS) in infected deep partial thickness burn wounds in which epidermis is lost completely and the entire papillary dermis and most of the recticular layer of the dermis is lost. Infection induced by inoculating 10(7) colony forming units (cfu) of Pseudomonas aeruginosa caused significant increase in wound size (20%) till day 15, which decreased significantly from day 9 by SSDM-CS treatment, showing complete healing by day 27 (control > or = 37 days). Early subsidence of infection (<10(2) cfu, day 9) by SSDM-CS resulted in faster epidermal resurfacing and fibroplasia, whereas heavy microbial load (>10(7) cfu, day 9) in controls caused severe inflammatory cellular infiltration. Persistent infection triggered early expression of proinflammatory cytokines intereukin-6, intereukin 1-beta, and tumor necrosis factor-alpha, lasting until day 9, whereas cytokine level decreased in SSDM-CS-treated group by day 6. Infection exacerbated expression of active matrix metalloproteinases (MMPs)-2 and -9 in controls (day 15), while SSDM-CS positively modulated MMP-2 and -9 with faster decline in their levels (day 12). Inherent nature of the dressing to maintain drug level at equilibrium therapeutic concentration (51.2 microg/mL) for prolonged time (72 h), below systemic toxic limits (20 microg/dL, serum level), accelerated the magnitude and sequence of reparative events.

Diseño de una matriz de soporte compuesta de colágeno de piel de tiburón-aloe para ingeniería tisular / Design of shark skin collagen-aloe composite scaffold for tissue engineering

Se ha demostrado que el colágeno es un nuevo biomaterial utilizado para la administración de fármacos, la fabricación de apósitos o como sustrato para ingeniería tisular cuya biocompatibilidad y propiedades biodegradables son únicas. El colágeno bovino y porcino tipo I constituyen una fuente fácilmente disponible de material de soporte para diversas aplicaciones biomédicas. Sin embargo, estas fuentes conllevan cierto riesgo potencial de enfermedades infecciosas como la encefalopatía espongiforme bovina o la encefalopatía espongiforme transmisible. Por esta razón, existe una demanda de colágeno tipo I procedente de otras fuentes. En el presente estudio, se utilizan animales acuáticos y, en concreto, especies de tiburón en las que el colágeno tipo I es una proteína principal de la piel y la estructura tiene similitud con la de las especies mamíferas. Se ha intentado utilizar colágeno de piel de tiburón como matriz de soporte con extracto de aloe para mejorar la estabilidad. Estas matrices de soporte se caracterizaron por varias propiedades fisicoquímicas y por la evaluación de biocompatibilidad para facilitar el crecimiento de fibroblastos dérmicos humanos in vitro. La incorporación de extracto de aloe influyó enormemente en la morfología y las propiedades fisicoquímicas de la matriz de soporte. Se observó in vitro que los fibroblastos conservaban la orientación organizada en forma de huso al cultivarse sobre la matriz de soporte de colágeno. Así, la matriz de soporte de colágeno desarrollada con una proporción de 10:1 de colágeno de piel de tiburón y extracto de aloe, respectivamente, sirvió como material biocompatible con una resistencia a la tracción apreciable. La investigación anterior sugiere que la matriz de soporte de colágeno de piel de tiburón desarrollada puede ser una alternativa efectiva al colágeno de mamífero en el campo de la ingeniería tisular y para diversas aplicaciones en la curación de heridas (AU)

Collagen has proven to be a novel biomaterial used for drug delivery, wound cover dressings or as a substrate for tissue engineering with unique biocompatibility and biodegradable properties. Bovine and porcine Type I collagen provide a readily available source of scaffold material for various biomedical applications. However these sources have some potential risk of infectious diseases such as bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Hence there is demand for an alternative Type I collagen from various other sources. The present study utilizes the aquatic animals particularly the shark species in which collagen Type I is a major protein in the skin and the structure has similarity to that of mammalian species. An attempt was made to use shark skin collagen as scaffold with the extract of aloe to improve the stability. These scaffolds were characterized for various physicochemical properties and biocompatibility assessment to support the growth of human dermal fibroblasts in vitro. The incorporation of aloe extract highly influenced the morphology and physicochemical properties of the scaffold. It was observed in vitro that the fibroblasts retained the spindle shape, organized orientation when cultured over collagen scaffold. Thus the developed collagen scaffold at 10: 1 ratio of shark skin collagen and aloe extract respectively served as a biocompatible material with appreciable tensile strength. The above investigation suggests that the developed shark skin collagen scaffold could be an effective alternative for the mammalian collagen for tissue engineering and various wound healing applications (AU)

Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: development and characterization.

Chronic wounds express elevated levels of proteases, in particular matrix metalloproteinases (MMPs), which degrades de novo granulation tissue and endogenous biologically active proteins. An effective therapeutic approach for chronic wounds would be to modify this hostile environment and reduce the proteolytic imbalance. Doxycycline has been proved recently to inhibit MMPs and used topically for chronic wound ulcers, beyond their antimicrobial profile. To this end, a carrier system for controlled release of doxycycline, suitable for incorporation into various wound dressings like membranes and sponges was developed. In the present study gelatin microspheres, cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was proposed. The cross-linking was carried out with different concentrations of EDC (10 mM, 50 mM and 100 mM) and for different time periods (3-24 h). The cross-linked microspheres were characterized by evaluating the extent of cross-linking, the morphology, swelling behaviour and drug loading and in vitro studies of drug release, enzymatic degradation and biocompatibility. The extent of cross-linking increased as a function of both EDC concentration and the cross-linking time periods. It is found that the extent of cross-linking greatly influences the swelling and drug release behaviour of the microspheres. The cross-linked microspheres were found to be biocompatible to NIH 3T3 mouse embryonic fibroblast. The overall study indicates that the zero length cross-linker EDC can be considered as a potential alternative for cross-linking the gelatin microspheres.

Design and delivery of silver sulfadiazine from alginate microspheres-impregnated collagen scaffold.

A reconstituted collagen scaffold impregnated with silver sulfadiazine (SSD) loaded alginate microspheres, capable of delivering the drug in a controlled manner has been developed. SSD-loaded alginate microspheres were prepared by modified water-in-oil emulsion technique through interfacial ionic gelation of alginate using CaCl2. The SSD-loaded microspheres were impregnated in pepsin-solubilized collagen, in situ, while inducing fibrillation and cast as thin scaffold. Morphological features of microspheres and microsphere-impregnated collagen were analyzed through SEM. Distribution homogeneity of impregnated microspheres, their in vitro behavior in (Dulbecco's modified minimal essential media) DMEM, and antibacterial efficiency against ATCC pathogens were determined. Initial drug load of 20% (w/w) with respect to alginate and 40% (v/v) of 2% alginate with respect to oil phase were found to produce microspheres of optimum drug entrapment (3%) and required size range (300-370 microm). In vitro drug release studies from the scaffold showed an initial burst release of 47.5% and a controlled release for 72 h with equilibrium concentration of 68.8%. SSD-loaded microspheres exhibited minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) levels of 32 and 40.2 microg/mL to both K. pneumoniae and E. coli respectively. P. aeruginosa showed MIC and MBC levels of 44.8 and 51.2 microg/mL respectively, while Staphylococcus aureus exhibited MIC and MBC at the same concentration range (57.6 microg/mL). The collagen-based scaffold impregnated with SSD-loaded alginate microspheres can deliver SSD in a controlled fashion, can control infection for extended time period with lesser dressing frequencies, and will enable easier assessment of wound.

Low molecular weight heparin-induced pharmacological modulation of burn wound healing.

Low molecular weight heparin (LMWH) appears to be a promising solution for reducing inflammatory post-burn episodes and results in improved healing. The clinical examination presented here includes patients with burn wounds ranging from 20 to 35% total body surface area (TBSA) who were categorized into two groups, of which one received subcutaneous LMWH treatment (10,000 units/day) and the other acted as control. The process of healing was assessed through regular examination of clinical features such as regression of erythema and oedema, eschar formation, and rate of re-epithelialization. Various studies have demonstrated an increase in levels of serum IL-6 indicating the severity of the morbid condition. In the present investigation, LMWH-treated patients exhibited a faster decline in levels of serum IL-6 (within 12 days) than control. Infiltration of inflammatory cells at the local wound site was assessed through a histological analysis of tissue samples taken on various days during the healing process. The LMWH-treated groups exhibited an organized healing pattern with better remodelling in a shorter duration (28 days), while control patients took more than 28 days for complete healing. A slight correlation was observed with TBSA to the inflammatory process, which subsided in patients treated with LMWH, favourably modulating the events involved in the inflammatory process of burn wound healing.

Cytotoxicity studies of chromium(III) complexes on human dermal fibroblasts.

The cytotoxicity of certain Cr(III) complexes, such as [Cr(salen)(H(2)O)(2)](+), [Cr(edta)(H(2)O)](-), [Cr(en)(3)](3+), [Cr(ox)(3)](3-), [Cr(pic)(3)], and CrCl(3), which differ in ionic character and ligand environment in human dermal skin fibroblasts, has been studied. After 72 h of exposure to 100 microM doses of chromium(III) complexes, the order in which the complexes had an inhibitory effect on cell viability was [Cr(en)(3)](3+) > [Cr(salen)(H(2)O)(2)](+) > [Cr(ox)(3)](3-) > [Cr(edta)(H(2)O)](-) > [Cr(pic)(3)] > CrCl(3). Based on viability studies it was confirmed that [Cr(en)(3)](3+), a triply charged cation, inhibits cell proliferation, and therefore, it was chosen to carry out further investigations. [Cr(en)(3)](3+), at a dose of 50 microM, was found to bring about surface morphological changes, evidenced by cellular blebbing and spike formation accompanied by nuclear damage. TEM analysis revealed substantial intracellular damage to fibroblasts in terms of the formation of apoptotic bodies and chromatin condensation, thus reflecting cell death. FACS analysis further revealed DNA damage by formation of a sub-G(1) peak with 84.2% DNA as aneuploid DNA and arrest of the G(2) / M phase of the cell cycle. Cellular DNA damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in DNA isolated from [Cr(en)(3)](3+)-treated fibroblasts. The proposed mechanism suggests the plausible role of Cr(V), formed as a result of oxidation of Cr(III) by cellular oxidative enzymes, in the cytotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the cytotoxicity and genotoxicity of a variety of Cr(III) complexes and to understand the potential hazards of Cr(III) complexes encountered in research laboratories.

In vivo and in vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn.

Petroleum ether extract of leaves and flowers of Hibiscus rosa-sinensis was evaluated for its potential on hair growth by in vivo and in vitro methods. In vivo, 1% extract of leaves and flowers in liquid paraffin was applied topically over the shaved skin of albino rats and monitored and assessed for 30 days. The length of hair and the different cyclic phases of hair follicles, like anagen and telogen phases, were determined at different time periods. In vitro, the hair follicles from albino rat neonates were isolated and cultured in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves and flowers. From the study it is concluded that the leaf extract, when compared to flower extract, exhibits more potency on hair growth.

Biocompatible collagen scaffolds from a human amniotic membrane: physicochemical and in vitro culture characteristics.

A reconstituted collagen membrane from human amnion has been investigated as a source of collagen matrix, which could be used as a substratum for culturing human fibroblasts. The suitability of pepsin-solubilized reconstituted human amniotic membrane, before and after cross-linking with chitosan, as a dermal matrix for culturing fibroblast was assessed by morphologic, physicochemical, cytotoxic and histochemical methods. Measurement of thermodynamic behaviour, by differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA), and tensile strength suggested that the cross-linked membrane had sufficient elasticity to serve as an efficient dermal substrate for in vitro culture of fibroblasts. Fibroblasts cultured on the chitosan cross-linked collagen membrane had good adherence, retaining their morphology as indicated by microscopic analysis. Proliferation of fibroblasts. observed on this membrane affirms its non-toxic nature. These results support the application of reconstituted human amniotic collagen membrane as collagenous scaffolds to culture fibroblasts in vitro.

Collagen-chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells.

A biodegradable polymer scaffold was developed using collagen and chitosan, in the form of interpenetrating polymeric network (IPN), for in vitro culture of human epidermoid carcinoma cells (HEp-2, Cincinnati). Glutaraldehyde was used as cross-linking agent for the development of scaffold. Various types of scaffolds were prepared using different proportionate mixtures of collagen and chitosan solutions in the ratio of 3:7, 4:6, 5:5, 6:4 and 7:3 (collagen:chitosan). These scaffolds were fully characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA). Equilibrium swelling studies were carried out in phosphate buffer of physiological pH (7.4) to study its swelling characteristics at slightly alkaline pH. The scaffold that showed optimum swelling property was selected as the best scaffold for performing in vitro culture studies. In vitro culture studies were carried out using HEp-2 cells, over the selected scaffold and its growth morphology was determined through optical photographs taken at different magnifications at various days of culture. The results of the above studies suggest that the scaffolds prepared from collagen and chitosan can be utilized as a substrate to culture HEp-2 cells and can also be used as an in vitro model to test anticancerous drugs.

Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli.

The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli.

Studies on the pathogenicity of classical enteropathogenic Escherichia coli strains isolated from acute diarrhoea among children 0-5 years of age.

The mechanisms of pathogenicity in EPEC strains were studied in tissue culture. Escherichia coli was isolated as the predominant organism in the primary culture of 1293 (70.54%) diarrhoeal cases. 284 (90.44%) cases from the age group of 1-6 months showed Escherichia coli as the predominant organism. Classical Enteropathogenic Escherichia coli) were detected in 311 (24.05%) cases. Among EPEC isolates 277 (89.06%) did not produce either LT or ST 32(10.28%) produced LT or ST. 2 strains produced verotoxins belong to serotypes 0:86; K:61, 0:26; K:60, sero groups 0.86 :K:61, 0.142:K 86, 0.128:K 67, 0.126:K 71, 0125:K 70 0119:K69 showed localised adherence and serogroups 0111:K58-055:K59 showed both localised and diffused adherence to HeLa cells.

A simple method to quantitate circulating immune complexes in different diseases.

Immune Complexes are involved in the Pathogenesis of many diseases of varied aetiology such as autoimmune disorders, protozoal diseases, bacterial and viral infections. Quantitation of immune Complexes in these diseases can be used for diagnosis and to ascertain the prognosis. The simple method of precipitation by polyethylene glycol and quantitation by single Radial Immunodiffusion has been used in leprosy, syphilis, bacterial endocarditis and systemic lupus erythematosus (SLE). This method found significantly higher levels of circulating immune complexes (CICs) in erythema nodosum leprosum, culture positive bacterial endocarditis and SLE where CICs are known to play an important role in the pathogenesis.

Differential diagnosis of viral hepatitis based on hepatitis viral markers.

192 patients of acute viral hepatitis (AVH) from three different hospitals of Madras metropolitan area during November 1985 to January 1986 were investigated for serologic markers of hepatitis A virus (anti HAVIgM) and hepatitis B virus (HBsAg, HBeAg, anti HBcIgM and anti HBs) by Enzyme linked immunosorbent assay (ELISA). While the overall pattern of AVH in Madras as revealed from the study showed Hepatitis A to be 36.4%, Hepatitis B 34.4% and Non-A Non-B 29.1%, the pattern differed significantly when areawise categorisation was done. The major AVH type in Government General Hospital was Hepatitis B (48.9%). While it was hepatitis A (46.9%) in Government Stanley Hospital and Non-A Non-B (40.0%) in Military Hospital. Using anti HBcIgM marker of Hepatitis B Virus and anti HAVIgM it was possible to make out that 13.5% of the cases, currently suffering from hepatitis A were either HBV carriers (8.3%) or cases convalescing from a previous Hepatitis B attack (5.3%). Various combinations of HBV markers positivity were observed and their diagnostic significance inferred.